The present invention concerns a mammalian cell line and its active fragments which when it is co-cultured with lymphocytes during which allogenic stimulation is avoided, activate lymphocytes to form tumoricidal T cells a process for the production of tumoricidal T lymphocytes by co-culturing lymphocytes with such cell lines or with active fragments thereof, the tumoricidal T lymphocytes obtainable by this process as well as the use of these T lymphocytes for the production of a therapeutic agent which can be used in tumour therapy.
The cellular immune defence plays an important role in the elimination of pathologically changed endogenic cells such as e.g. cells infected by viruses or tumour cells. In this process cytotoxically active T lymphocytes recognize the changed endogenic cells on the basis of surface antigens. These surface antigens are usually protein fragments which are formed by the cells and are present on the cell surface bound to surface receptors of the so-called major-histocompatibility complex (MHC) (Zinkernagel et al., Nature 248 (1974), 701-702 and Babbit et al., Nature 317 (1985), 359-361). However, if these surface antigens of the tumour cells only differ slightly from the corresponding antigens of healthy cells, the immune system may possibly form no cytotoxically active T lymphocytes which could eliminate the tumour cells.
Therefore attempts have already been made to induce a cellular immune resistance against such tumour cells. For this it was firstly attempted to achieve an active immunization with unspecific immunostimulants such as Bacillus Calmette-Guerin (BCG), Corynebacterium parvum or vaccines from tumour cell extracts (Terry and Rosenberg eds., Immunotherapy of Human Cancer (1982), Elsevier North Holland). Better results were obtained using the concept of so-called adoptive immunotherapy. In this case lymphocytes of the patient are activated in vitro and then re-implanted. The in vitro activation to form such xe2x80x9cpromiscuous killer cellsxe2x80x9d (D. Thiele et al., Immunology Today 10 (1989), 375-381) is usually carried out by addition of interleukin 2. The cytotoxic lymphocytes obtained are then denoted lymphokine-activated killer cells (LAK cells) (Rosenberg, Immunology Today 9 (1988), 58-62). In contrast to cytotoxic T lymphocytes, the action of LAK cells against tumour cells does not depend on a correct expression of the MHC genes for the recognition of tumour antigens and in contrast to the natural killer cells of the immune system LAK cells are also effective against fresh tumour cells. It has even already been possible to achieve the first clinical successes using LAK cells. However, a disadvantage of this form of adoptive immunotherapy are side-effects of interleukin 2 which is required in relatively high doses over a longer time period. This results primarily in an increase in the permeability of the capillaries and concomitant functional disorders of the organs (Rosenberg, Immunology Today 9 (1988), 58-62, Rosenstein et al., Journal Immunology 137 (1986), 1735-1742 and Ettinghausen et al., Surg. Forum 37 (1987), 388-389). In addition such LAK cells are obtained which when stimulated with interleukin 2, are directed against healthy endogenous cells (B. Chen et al., Cell. Immunol. 118 (1989), 458-469).
In the search for more effective methods for adoptive immunotherapy the lymphocytes to be activated were also cultured in the presence of autologous tumour cells (mixed lymphocyte tumor cultures, G. Fossati et al., International Journal of Cancer 42 (1988), 239-245; G. Degiovanni et al., Eur. J. Immunol. 18 (1988), 671-676; Wxc3x6lfel et al., J. Exp. Med. 170 (1989), 797-819; Darrow et al., J. Immunol. 142 (1989), 3329-3335 and Notter et al., Int. J. Cancer 45 (1990), 834-841). In addition a method for the proliferation of tumour-infiltrating lymphocytes (TIL) in vitro has also been described (Yron et al., J. Immunol. 125 (1980), 238-245) in contrast to LAK cells, these tumour-infiltrating lymphocytes have a high tumour specificity i.e. they are only active against the tumour from which they themselves were isolated. Such tumour-infiltrating lymphocytes are not even effective against the same type of tumours from other patients. This significantly limits their therapeutic applicability.
The object of the present invention was therefore to provide tumoricidal T lymphocytes which are more suitable for tumour therapy than the previously known in vitro activated T lymphocytes.
This object is achieved by a mammalian cell line or active subcellular fractions thereof which are characterized in that
a) when they are co-cultured with lymphocytes during which allogenic stimulation is avoided they activate lymphocytes to form tumoricidal T cells without the need to add mitogens or growth factors such as e.g. interleukin 2 and
b) the lymphocytes activated in this way proliferate in their presence without addition of interleukin 2.
Surprisingly it turned out that tumoricidal T lymphocytes with a broad tumoricidal activity without HLA restriction can be obtained from lymphocytes by co-culture with a cell line according to the present invention or active subcellular fractions/fragments thereof. In this connection tumoricidal activity is understood as a killing effect and in particular a lytic effect on the respective tumour cells as well as an inhibitory action on the proliferation of these tumour cells.
A cell line is understood as those cells which have the capability of unlimited proliferation such as is characteristic of HeLa cells (ATCC CCL 2) (James D. Watson et al., Molecular Biology of the Gene, 4th edition, The Benjamin/Cummings Publishing Co., Inc. (1987), p. 963). Such cell lines are for example obtained by immortalizing human blood lymphocytes. Immortalization is preferably carried out by fusion with cytoplasts from the mouse myeloma cell line Ag8.653 according to the method described in EP-B 0 093 436 or in EP-B 0 256 512 (the content of which is also subject matter of the present patent application). The immortalized lymphocyte lines thus obtained are then co-cultured with human donor lymphocytes.
Blood lymphocytes are preferably used as lymphocytes. However, it is also possible to use tumour-infiltrating lymphocytes (TIL) as well as lymphocytes from the spleen or lymphatic nodes. In this connection it is preferable to purify the lymphocyte preparation before use. When using blood lymphocytes it is particularly expedient to substantially remove the erythrocytes and to concentrate the mononuclear cells. It is also advantageous to deplete the number of cells which can be allogenically stimulated by the cell line according to the invention or active fragments thereof.
In order to avoid allogenic stimulation, lymphocytes which are susceptible to such stimulation are eliminated from the donor lymphocyte population before co-culture. For this monocytes, macrophages, natural killer cells and MHC-restricted cytotoxic T cells, also especially those directed against allogenic MHC of the activator cell line and their precursor cells are preferably eliminated by incubation with L-leucyl-L-leucine methyl ester according to Thiele and Lipsky (The Journal of Immunology, Vol. 136, No. 3 (1986), p. 1038-1048). Those immortalized lymphocyte lines are selected after the co-culture which cause an activation of the donor lymphocytes to form tumoricidal T lymphocytes during this co-culture. In this process those activating lymphocyte lines which are lysed by the donor lymphocytes which activate them during the co-culture are preferably examined further. For the further selection, these activating lymphocyte lines are cultured together with the donor lymphocytes which are activated by them and a series of different tumour cell lines. Finally those activating lymphocyte lines are selected which lead to activated donor lymphocytes with tumoricidal action against the examined tumour cell lines. In this case tumoricidal action is not only to be understood as the killing, in particular lysis, of the examined tumour cell lines but also an inhibitory effect on proliferation. This tumoricidal action can for example be detected by means of cytotoxicity tests familiar to a person skilled in the art, for example in that the tumour cell lines which can be distinguished morphologically from the tumoricidal T lymphocytes as well as from the activating lymphocyte line disappear from the co-culture or are at least overgrown by tumoricidal T lymphocytes during longer culture. The tumoricidal T lymphocytes are preferably CD3+, CD4+and/or CD8+.
A preferred subject matter of the present invention is a lymphocyte cell line, particularly preferably a B lymphocyte cell line, or active fragments thereof, which is characterized in that
a) when they are co-cultured with lymphocytes in which allogenic stimulation is avoided they activate lymphocytes to form tumoricidal T cells without having to add mitogens or growth factors such as e.g. interleukin 2 and
b) the lymphocytes activated in this way proliferate in their presence without addition of interleukin 2.
The human B cell lines HB 654 and HB 617 are especially preferred.
Using the cell lines and active fractions according to the present invention it is possible by simple co-culture with lymphocytes while avoiding allogenic stimulation to cause an activation of these lymphocytes to form tumoricidal T cells. This activation preferably takes place while the lymphocytes are in direct contact with the cell lines or active fractions (fragments) thereof. It has turned out that addition of antibody against IL2 and/or the IL2 receptor inhibits the activating effect of the cell lines according to the invention.
Therefore the present invention also concerns a process for the production of tumoricidal T lymphocytes by co-culturing lymphocytes with a cell line according to the present invention or active fragments thereof.
In order to carry out the process according to the present invention lymphocytes (preferably mononuclear lymphocytes) are firstly isolated from the blood or from tumours of a donor according to known methods e.g. by a Ficoll density gradient centrifugation. Subsequently the remaining lymphocytes are cultured in the usual lymphocyte culture medium together with a cell line according to the present invention or active fragments thereof, preferably the human B cell lines HB 654 and/or HB 617 under conditions which enable cell contact. In this process the cell line is preferably added to the lymphocytes in a deficit of 1:100. The culture is continued until the activation of tumoricidal T cells can be detected on the basis of the elimination of the activating cell line. A culture of about 8 days is usually necessary for this. An activation and proliferation of tumoricidal T lymphocytes is achieved by means of the co-culture according to the present invention without having to add growth factors or mitogens such as e.g. lymphokines, in particular interleukin 2. This is particularly important for the therapeutic use of the tumoricidal T lymphocytes obtained since such factors can produce side effects during the therapeutic application. However, a persistent proliferation of the tumoricidal T lymphocytes obtained requires the constant presence of the cell line according to the present invention or active fragments thereof and the possibility of forming cellxe2x80x94cell contacts. Since the tumoricidal activity of the activated lymphocytes obtained is also directed against the cell line according to the present invention, it is therefore necessary to continuously supply this cell line in order to achieve persistent proliferation. Although, without such an addition proliferation of tumoricidal T lymphocytes stagnates after one to two days, the tumoricidal T lymphocytes survive for three to four weeks during which they transform from blasts into very small cells which join together to form aggregates. They retain their tumoricidal activity and can again be converted into a proliferating state after a latency period of three to six days by addition of the cell line according to the present invention or active fragments thereof.
In addition to vital proliferable cells for co-culturing with the cell line according to the present invention it is also possible to use a cell line according to the present invention treated with mitomycin which has been lethally irradiated or chemically immobilized e.g. with formaldehyde or a subcellular fraction such as e.g. a membrane fraction, membrane vesicle or an extract from such a subcellular fraction. Furthermore the cell line according to the present invention can also be fused with other cells and the fusion cells obtained can be used for activation.
Therefore the present invention also concerns a process for the production of tumoricidal T lymphocytes by co-culturing lymphocytes from blood, during which allogenic stimulation is avoided, with a cell line according to the present invention, active derivatives or subcellular fractions of the cell line according to the invention or with fusion products of this cell line with other cells without adding mitogens or growth factors such as interleukin 2.
Since growth factors or mitogens do not have to be added in such a process for the production of cytotoxic T lymphocytes and interleukin 2 does not have to be added to the tumoricidal T lymphocytes obtained in order to continue their proliferation, they are better suited for an application in tumour therapy than the previously known promiscuous killer cells such as e.g. LAK cells. Due to their broader tumoricidal activity they are better suited for therapeutic application than tumour-infiltrating lymphocytes. In contrast to natural killer cells the tumoricidal cells produced according to the process according to the present invention are T cells.
The present invention therefore also concerns tumoricidal T lymphocytes having a broad tumoricidal activity which are characterized in that
a) they have a tumoricidal effect on the tumour cell lines MOLT-4, Jurkat, THP-1, HL-60, HeLa, K-562, Malme-3M and Y79 and
b) no interleukin 2 is detectable in the culture supernatant of these tumoricidal T lymphocytes during proliferation of these cells in the presence of the cell line HB 654 or HB 617 at a detection limit of 0.5 IU/ml.
The tumoricidal T lymphocytes according to the present invention are thus obtainable by simply co-culturing lymphocytes with a cell line according to the present invention or active derivatives or subcellular fractions of this cell line on a fusion product of this cell line with another cell until the activation of lymphocytes to tumoricidal T cells is detectable e.g. by means of the elimination of the activating cell line. Surprisingly it turned out that the tumoricidal T lymphocytes obtained in this way have a tumoricidal action on a multitude of tumour cell lines such as e.g. MOLT-4, Jurkat, THP-1, HL-60, HeLa, K-562, Malme-3M and Y79. A further distinguishing feature of these tumoricidal T lymphocytes is that interleukin 2 cannot be detected in their culture supernatant neither during the activation nor during subsequent proliferation of the activated cells (IL2 ELISA; DuPont, Catalogue No. NEK-057; lower detection limit 0.5 IU/ml).
As already stated human tumoricidal T lymphocytes are obtainable using the activating cell line according to the present invention without having to add mitogens or growth factors such as lymphokines, in particular interleukin 2, which can lead to side effects during therapy. Thus the present invention also concerns the use of tumoricidal T lymphocytes according to the present invention for the production of a therapeutic agent which can be used in tumour therapy. For such a therapeutic application the tumoricidal T lymphocytes according to the present invention are washed according to methods known to a person skilled in the art (e.g. by centrifugation and resuspension of the pellet in physiological saline which is repeated several times e.g. three times), they are isolated if desired and taken up in a medium suitable for the administration (e.g. physiological saline).
In addition to this ex vivo activation of lymphocytes to tumoricidal T lymphocytes, lymphocytes can also be activated in vivo to tumoricidal T lymphocytes by administration of an activating cell line according to the present invention or derivatives or subcellular fractions of this cell line. The activating cell line is washed according to methods known to a person skilled in the art for such a therapeutic application and taken up in a medium suitable for the administration such as e.g. physiological saline.
Therefore the present invention in addition concerns the use of an activating cell line according to the present invention or an active subcellular fraction (fragments) or appropriate derivative of this cell line which induces lymphocytes to form tumoricidal T cells for the production of a therapeutic agent which is applicable in tumour therapy.
The active subcellular fractions are particularly suitable for a direct in vivo application. These fractions can be used to directly activate lymphocytes in the body to tumoricidal T lymphocytes. It is particularly advantageous to apply these fractions directly to the tumour in order to activate tumour-infiltrating lymphocytes to tumoricidal T lymphocytes.
An active subcellular fraction is understood as a fraction of a cell line according to the invention which induces lymphocytes to form tumoricidal T cells in an analogous manner to the cell lines according to the invention (e.g. HB 617 and HB 654). Such fractions can for example be subcellular vesicles that are obtained by hypotonic shock or cell-free membrane vesicles that are obtained by incubation with Cytochalasin B. An eluate from the cell lines according to the invention that can for example be obtained after incubation with sodium chloride and sodium citrate is also suitable. Such fractions of the cell lines according to the invention can be purified further by methods familiar to a person skilled in the art, for example by chromatographic purification during which the activity of the fraction (the property of forming tumoricidal T lymphocytes) has to be checked after each purification step.
The invention therefore also concerns a process for producing an active fraction from lymphocyte cell lines which
a) activates lymphocytes to tumoricidal T cells in a co-culture with lymphocytes in which an allogenic stimulation is avoided without having to add mitogens or growth factors and
b) the lymphocytes activated in this manner proliferate in its presence without the addition of interleukin 2
xe2x80x83which is characterized in that a mammalian cell line which has these properties
(i) is fractionated
(ii) the fractions are separated and it is examined whether these fractions activate lymphocytes to tumoricidal T cells in an analogous manner to the initial cell line,
(iii) such an active fraction is selected and is further fractionated and isolated while checking its activity until the desired degree of purity has been reached.
The tumoricidal T lymphocytes can also be used ex vivo to eliminate tumour cells in a cell preparation. This can preferably be used to eliminate (purging) tumour cells from stem cell isolates (e.g. bone marrow stem cells) by co-culture with tumoricidal T lymphocytes or a lymphocyte cell line according to the invention. The stem cells purified in this manner can for example again be implanted in the patient after radiation or chemotherapy (autologous bone marrow transplantation).
Finally, the corresponding therapeutic compositions are also a subject matter of the present invention which contain tumoricidal T lymphocytes according to the present invention or an activating cell line according to the present invention or a subcellular fraction or corresponding derivative of this cell line which induces lymphocytes to form tumoricidal T cells, in each case together with the usual pharmaceutical carrier, filling and/or auxiliary agents.
The cell line HB 654 according to the invention was deposited on 24.03.1993 at the xe2x80x9cDeutsche Sammlung fxc3xcr Zellkulturen und Mikroorganismen GmbHxe2x80x9d, Mascheroder Weg 1 b, D-3300 Braunschweig under the number DSM ACC 2122.
The cell line HB 617 according to the invention was deposited on 11.03.94 at the xe2x80x9cDeutsche Sammlung fxc3xcr Zellkulturen und Mikroorganismen GmbHxe2x80x9d, Mascheroder Weg 1 b, D-3300 Braunschweig under the number DSM ACC 2166.
The present invention is elucidated further by the following examples.